1. Field of the Invention
The invention relates to the process for preparation of protoescigenin from escin.
Protoescigenin is a polyhydroxy aglycone obtained during the hydrolysis of escin, the natural saponin present in Aesculus hippocastanum seeds. Protoescigenin may be a valuable substrate used in the synthetic modification and derivatization of the naturally occurring saponins.
2. Description of the Related Art
The mixture of saponins known under the common name escin, is the major component of Aesculus hippocastanum seeds extracts. It is present in the three forms named α-escin, β-escin and cryptoescin. These saponins belong to polyhydroxy triterpene glycosides, containing four different aglycones (sapogenins), such as escigenin, protoescigenin, barringtogenol C and barringtogenol D, which are characterized by different substituents at the C-16/C-21 and the C-24 hydroxyl groups.
Until now 79 saponins have been isolated and characterized from the hydrolyzates of Aesculus hippocastanum seeds extracts. Most of these compounds consist of a trisaccharide chain containing glucuronopyranosyl residue linked via a glycosidic bond to the C-3 atom of an aglycone, acyl groups at the C-21, C-22 and C-28, and seldom at the C-16 position. Acyl moieties embrace angeloyl, tigloyl, acetyl, 2-methylbutanoyl and 2-methylpropanoyl groups (Pharmaceutical Crops, 2010, 1, 24-51).
The extracts of Aesculus seeds usually vary in composition and the difference depends on a plant species as well as the origin of a plant growth. The chemical composition of saponins isolated from the horse chestnut Aesculus hippocastanum L. seeds, growing predominantly in Europe and North America was proposed by R. Tschesche, et al. in Justus liebigs Ann. Chem., 669, 171-182(1963) and can be illustrated by the structure shown in FIG. 6.
The research of Yoshikawa and co-workers resulted in the isolation and identification of 12 saponins, which proved to be the main components of Aesculus hippocastanum seeds extracts. The outcome of these works was published inter alia in Chem. Pharm. Bull. 42(6), 1357-1359 (1994); Chem. Pharm. Bull. 44(8), 1454-1464 (1996); Biol. Pharm. Bull. 20(10), 1092-1095 (1997), Chem. Pharm. Bull. 46(11), 1764-1769 (1998). The identified compounds included escin Ia, Ib, IIa, IIb, IIIa, IIIb, IV, V and VI and also isoescin Ia, Ib, and V, the chemical structures of which are summarized in the table below.
NameAglyconeR1R2R3R4R5R6Escin IaPESHTigAcOHHGlc-pEscin IbPESHAngAcOHHGlc-pEscin IIaPESHTigAcOHHXyl-pEscin IIbPESHAngAcOHHXyl-pEscin IIIaBACHTigAcHHGal-pEscin IIIbBACHAngAcHHGal-pEscin IVPESHAcAcOHHGlc-pEscin VPESHMPAcOHHGlc-pEscin VIPESHMBAcOHHGlc-pIsoescin IaPESHTigHOHAcGlc-pIsoescin IbPESHAngHOHAcGlc-pIsoescin VPESHMPHOHAcGlc-pProtoescigeninPESHHHOHH—Barringtogenol CBACHHHHH—PES—protoescigeninBAC—barringtogenol C
Escin, due to its beneficial effects on venous tone, anti-inflammatory and anti-edemic activity, is wildly used in medicine, mainly in the treatment of chronic venous insufficiency, but it found use in the cosmetics as well. Although the efficiency of escin has been proved in the traditional medicine as well as in the clinical treatment, the molecular basis of its activity has not been established yet. The complexity of the saponin mixture and the lack of validated analytical methods, necessary for a qualitative and a quantitative determination of the natural compounds composition, impedes studies of pharmacokinetic and biochemical mechanism.
Isolation of the individual saponins from the crude plant material, determination of their structure and their analysis require application of laborious and advanced analytical techniques. These difficulties result from the unique and complex chemical structures of saponins, the similarity of their structure and resulting similar physicochemical properties, for example polarity, and in the end lack of chromophores, which hinders detection of analyzed molecules.
In general, saponins are isolated from the crude plant material by extraction with the mixture of water and an alcohol, such as methanol or ethanol, followed by evaporation of the solvents under reduced pressure, reconstitution of the residue in a small amount of water and separation between n-butanol/water diphase system. For further purifications, the column chromatography techniques or liquid-liquid chromatography separation method are employed, but usually high-performance liquid (HPLC) chromatography must be used. In most cases, obtaining the high purity saponins requires multiple chromatographies, which involves the replacement of column filling and the change of eluting solvents.
For instance, according to the procedure published in Chem. Pharm. Bull. 44(8), 1454-1464 (1996), the crude methanolic extract of Aesculus hippocastanum L. seeds was chromatographed on Diaion HP-20 column, followed by another separation of methanolic fraction of saponins mixture in reversed phase, on the chromatography column filled with silica gel. Multiple HPLC chromatographies of 90% methanolic eluate containing the pre-purified mixture of saponins furnished separation of escin Ia, Ib, IIa, IIb and IIIa.
In Justus Liebigs Annalen der Chemie 1963, 669, 183-188 Kuhn R. and Loew I. described the hydrolysis of escin in the solution of 4 N hydrochloric acid in ethanol, the separation of the intermediate, and its subsequent hydrolysis under basic conditions with potassium hydroxide in methanol. The hydrolysis products were separated by chromatography on silica gel, yielding protoescigenin and escigenin.
Yoshika I. et al. separated and determined the chemical structure of sapogenins isolated from Japanese Aesculus turbinata BLUME extract. They also assigned the configuration of carbon atoms bound to hydroxyl groups in ring E of protoescigenin. These findings were published in Chem. Pharm. Bull. 19(6), 1200-1213 (1971). According to their procedure, n-butanolic extract of Aesculus seeds was condensed in vacuo furnishing a resin residue. This product was refluxed in ether resulting in precipitation of the solid of the crude mixture of saponins, which was subsequently hydrolyzed in 4 N hydrochloric acid in ethanol at elevated temperature. The obtained mixture was diluted with water, condensed and diluted with water again to give a solid precipitate, which was hydrolyzed in the basic medium with 5% KOH methanolic solution. After water addition, the crude mixture of sapogenins precipitated out of the solution, the solid was purified by chromatography on the column filled with aluminum oxide and yielded a mixture of four main compounds. The major sapogenin was purified by crystallization in methanol and precipitated into colorless needles, characterized by a 300-307° C. melting point. The physicochemical data, such as the melting point, IR (KBr) spectra and TLC analyses were consistent with the structure of protoescigenin.
According to the publications identified above, cleavage of the glycosidic bond of deacylated escin occurs during the hydrolysis under acidic conditions. In this process, protoescigenin (deacylated escin II methanolysis), and barringtogenol (deacylated escin III methanolysis) are formed. Under the basic conditions hydrolysis of saponin acyl groups takes place, liberating the molecules of acetic, tyglic and angelic acids.
Although different methods of hydrolysis are well known to those skilled in the art, methods other than chromatography separation of the products of saponin hydrolysis have not been found in the prior art. In all the procedures described in the publications mentioned above, hydrolysis is preceded by chromatographic purification of either the mixtures or individual saponins. Following these multi-step purification processes, some of the pentacyclic triterpenes were successfully isolated and purified on laboratory scale. However, these elaborate and expensive methods cannot be implemented on industrial scale.
The main problem one must face while scaling-up the process, is the viability of the crude extract composition, the similar polarity, and the similarity of molecular weight of the particular saponin mixture components. These physicochemical properties of the crude mixture, as well as the lack of standardization methods designed for the plant raw materials and products, impede the separation of individual saponins either by crystallization or ultrafiltration. Possibility of obtaining protoescigenin of high purity and at bulk quantities is crucial, for the substrate to be used in synthetic modifications. In the molecule of protoescigenin six hydroxyl groups are present. Thus the number of possible products resulting from substitution of hydroxyl groups amounts to 63 (26−1). This number may dramatically increase if protoescigenin is contaminated with aglycones of other saponins. In the aftermath of chemical reaction, complex mixtures of products of similar structures are formed, the separation of which is impossible.
The results of experimental attempts to produce protoescigenin by the hydrolysis of escin demonstrate that the reaction product is usually the mixture of sapogenins, containing from 40 to 60% of protoescigenin only, as determined by HPLC. Among the other main components of the mixture, barringtogenol C was also detected, accompanied by the smaller amounts of other sapogenins, such as escigenin and barringtogenol D. Methods routinely used for the purification and isolation, for example the multiple crystallization, liquid-liquid or liquid-solid extractions, were not successful in the protoescigenin isolation.